Washington University in St. Louis, Department of Biomedical Engineering, St. Louis, MO 63130, USA.

Abstract

In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.

In vivo en face photoacoustic images of the skin of mouse ears in the form of maximum amplitude projection (MAP). The images were acquired by using wavelengths of 240, 245, 248, 250, 251, 252, 255, 260, 266, 270, 275, and 280 nm, respectively. Cell nuclei in the mouse skin are shown in the images at 245, 248, 250, 251, 252, 255, 260, 266, 270, and 275 nm but are unidentifiable in the images at 240 and 280 nm. PA: photoacoustic.

In vivo en face distribution of cell nuclei in the skin of mouse ears. (a) In vivo MAP photoacoustic image of cell nuclei distributed in mouse skin, acquired at a wavelength of 250 nm. PA: photoacoustic. (b) Histogram of the nuclear diameter (). Bin width is 0.6 μm. The solid curve is a Gaussian fit with a mean of 8.6 μm and a standard deviation (SD) of 1.6 μm (coefficient of determination ). (c) Histogram of the internuclear distance (). Bin width is 2 μm. The solid curve is a Gaussian fit with a mean of 22.7 μm and a SD of 3.6 μm ().

Signal and contrast of in vivo photoacoustic images of cell nuclei versus the optical wavelength. (a) Plot of SNR () of nuclear images versus wavelength. The SNR was collected from 25 cell nuclei in MAP photoacoustic images for each wavelength. (b) Plot of CNR () of nuclear images versus wavelength. The mean was calculated from 25 nuclei. (c) Typical absorption spectra of DNA and protein. The DNA is thymus DNA, and the protein is glutamate dehydrogenase. The absorption spectrum of DNA is adapted from Kunitz (1950) and Sambrook et al. (2001), and that of protein from Olson et al. (1952) and Gill et al. (1989). (d) The ratio of DNA to protein, defined as the ratio of the absorption coefficient of thymus DNA to that of glutamate dehydrogenase.

In vivo photoacoustic imaging of cell nuclei in the skin of mouse ears with varied laser pulse energy at a wavelength of 250 nm. (a) In vivo MAP photoacoustic images of cell nuclei at pulse energies of 10, 3, and 2 nJ. PA: photoacoustic. (b) CNR () of the nuclear images. The CNR was collected from 25 cell nuclei in MAP photoacoustic images for each pulse energy.